Abstract
The application of synthetic messenger RNA (mRNA) exhibits various advantages, such as expression of desired proteins in cells without genomic integration. In the field of tissue engineering, synthetic mRNAs could be also used to modulate the protein expression in implanted cells. Therefore, in this study, we incorporated synthetic humanized Gaussia luciferase (hGLuc) mRNA into alginate, chitosan, or chitosan-alginate hybrid hydrogels and analyzed the release of hGLuc mRNA from these hydrogels. After 3 weeks, 79% of the incorporated mRNA was released from alginate hydrogels, approximately 42% was released from chitosan hydrogels, and about 70% was released from chitosan-alginate hydrogels. Due to the injectability, chitosan-alginate hybrid hydrogels were selected for further investigation of the bioactivity of embedded hGLuc mRNA and the stability of these hydrogels was examined after the incorporation of synthetic mRNA by rheometric analysis. Therefore, HEK293 cells were incorporated into chitosan-alginate hydrogels containing mRNA transfection complexes and the luciferase activity in the supernatants was detected for up to 3 weeks. These results showed that the biodegradable chitosan-alginate hybrid hydrogels are promising delivery systems for sustained delivery of synthetic mRNAs into cells. Since chitosan-alginate hybrid hydrogels are injectable, the hydrogels can be simultaneously loaded with cells and the desired synthetic mRNA for exogenous protein synthesis and can be administered by minimally invasive local injection for tissue engineering applications.
Highlights
In recent years, the application of synthetic messenger RNAs for the production of therapeutic proteins has gained interest due to their advantageous properties over the use of DNA-based methods, including a safe, easy, and efficient translation of proteins [1,2]
After 3 weeks, 79% of the incorporated messenger RNA (mRNA) was released from alginate hydrogels, approximately 42% was released from chitosan hydrogels, and about 70% was released from chitosan-alginate hydrogels
Chitosan-alginate hybrid hydrogels were selected for further investigation of the bioactivity of embedded humanized Gaussia luciferase (hGLuc) mRNA and the stability of these hydrogels was examined after the incorporation of synthetic mRNA by rheometric analysis
Summary
The application of synthetic messenger RNAs (mRNAs) for the production of therapeutic proteins has gained interest due to their advantageous properties over the use of DNA-based methods, including a safe, easy, and efficient translation of proteins [1,2]. Alginate solutions can be injected in vivo and hydrogels can be generated by Ca2+ ions that are present in the surrounding tissue [39] Due to their similarity to extracellular matrix proteins, alginate hydrogels have been used as a carrier for cells, and have proven to promote the release of nucleic acids, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) [40,41,42,43]. Thereby, injectable chitosan solutions can be generated, which are thermo-responsive at physical pH and enable in situ formation of gels upon warming to body temperature [44] Due to their charge, cationic polymers are favorable for delivery of anionic molecules. Afterwards, the bioactivity of incorporated mRNA and the transfectability of cells were determined
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